Real time monitoring of mammalian circadian rhythm by bio-sensor

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It is considered that protein assembly on solid substrates is the important key technology to fabricate high performance biosensing systems and bioelectronics devices. The purpose of this study is to develop novel protein assembling method using bioaffinity binding and dissociation. The author paid attention to bioaffinity binding reaction between biotin analogues and avidin molecule. It is expected that biotin analogue-modified protein molecules are assembled on the solid supports by using avidin molecules as binders and that the constructed protein complexes are decomposed by the exchange reaction with free biotin molecules. This assembling technique might be available to fabricate recycle usable biosensing system and intelligent bioreactor system in which biocatalysis is controlled by the addition of ligand molecules as stimulation signal.Bioaffinity binding and dissociation reactions of avidin and biotin- or biotin analogue-modified proteins were characterized with evanescent wave- … More exciting fluorescence measurement. Dissociation of avidin and biotin analogue-modified protein complexes was certainly induced by the free biotin addition. It was demonstrated that biotin sensing is possible in the concentration range from 1x10^<-9> M to 1x10^<-5> M by the measurement of dissociation degree. The data further suggested that the biotin concentration for dissociation induction is dependent on the bioaffinity between the biotin analogue and avidin.At second, It was demonstrated that redox coenzyme-modified avidin molecules were assembled on the biotin-modified electrode surface and functioned as bioelecfrocatalyst for NADH oxidation. Dissociation of themodified avidin from the biotin-modified electrode surface was also induced by biotin addition.During three years we cloned putative mammalian clock genes (mPer1, mPer2, mPer3 and mTim). mPer1-promotor-Luciferasefusion gene was transfected to the Rat1 fibroblast cells. We observed the change of luminescence at single cell level by a sensitive photon detector. After serum shock, the luciferin luminescence begins to show the rhythm in a period length between 20 to 28 hours. Less